Methods and devices for micro-isolation, extraction, and/or analysis of microscale components in an array

ABSTRACT

Provided herein are devices and methods for the micro-isolation of biological cellular material. A micro-isolation device described can comprise a photomask that protects regions of interest against DNA-destroying illumination. The micro-isolation device can further comprise photosensitive material defining access wells following illumination and subsequent developing of the photosensitive material. The micro-isolation device can further comprise a chambered microfluidic device comprising channels providing access to wells defined in photosensitive material. The micro-isolation device can comprise a chambered microfluidic device without access wells defined in photosensitive material where valves control the flow of gases or liquids through the channels of the microfluidic device. Also included are methods for selectively isolating cellular material using the devices described herein, as are methods for biochemical analysis of individual regions of interest of cellular material using the devices described herein. Further included are methods of making masking arrays useful for the methods described herein. The micro-isolation devices can comprise a unique combination of barcodes in each microfluidics well, allowing two-dimensional mapping of genetic information.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation application of Ser. No.14/996,613 filed on Jan. 15, 2016 which, in turn, claims priority toU.S. Provisional Application 62/104,609 filed on Jan. 16, 2015, and maybe related to U.S. patent application Ser. No. 14/511,778 filed on Oct.10, 2014, and U.S. Pat. No. 8,889,416 issued on Nov. 18, 2014, all ofwhich are incorporated herein by reference in their entirety.

STATEMENT OF GOVERNMENT GRANT

This invention was made with government support under Grant No. CA174416awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD

The present disclosure relates to micro-isolation of microscalecomponents such as tissue and/or cell samples. More specifically, itrelates to methods and devices for such micro-isolation in an array.

BACKGROUND

The isolation of certain microscale components is an important factor inseveral applications where the ability to differentially analyzeproperties exhibited by varying types of components (e.g. cell types) isdesired.

For example, the ability to recognize properties typical of a componentincluded in a matrix with other similar components, can be of importancein various fields, including in particular biological fields. Inparticular, the ability to identify properties which cause a cell tobehave in a certain way is expected to promote an understanding of howcells behave both normally and abnormally. For example, the ability toselectively analyze cancerous cells is expected to provide insight intothe particular biochemical activities of those cells relative to normalcells.

However, separating different cell types and in particular cancerouscells from non-cancerous cells can be a difficult endeavor.

SUMMARY

Provided herein are apparatuses and methods for the micro-isolation ofmicro-scale components such as cellular material, which in severalembodiments provide for the selective biochemical analysis of desiredcomponents.

According to a first aspect, a method is described, the methodcomprising: a) fabricating a distributor chip comprising: a plurality ofmicrofluidics channels, each microfluidics channel having an input porton a first surface and a plurality of output ports on a second surfaceopposite the first surface, each microfluidics channel, its input portand its plurality of output ports laying on a plane parallel to asubsequent or previous microfluidics channel, thereby obtaining parallelrows of the output ports on the second surface; b) fabricating a firstoverlay comprising parallel rows of microfluidics wells having positionscorresponding to the parallel rows of the output ports; c) fabricating areceiver chip comprising parallel rows of microfluidics conduits havingpositions corresponding to the parallel rows of the microfluidics wellsof the first overlay; d) fabricating a stopper layer permeable to airand impermeable to water; e) providing a support layer comprisingparallel rows of openings corresponding to the parallel rows of themicrofluidics conduits of the receiver chip; f) positioning thedistributor chip on the first overlay, the first overlay on the receiverchip, the receiver chip on the stopper layer, and the stopper layer onthe support layer; g) aligning the distributor chip, the first overlay,the receiver chip, the stopper layer and the support layer, by aligningthe parallel rows of the output ports, the parallel rows of themicrofluidics wells, the parallel rows of the microfluidics conduits,and the parallel rows of openings, thereby creating an aligned assembly;h) inserting a solution in each input port of each microfluidics channelof the plurality of microfluidics channels, each solution comprisingsame reagents and a different barcode for each input port; and j)removing the first overlay from the aligned assembly, thereby obtainingparallel rows of microfluidics wells, each row having the same reagentsand a unique barcode in each row of the parallel rows of microfluidicswells of the first overlay.

According to a second aspect, a method of barcoding a sample having atarget sequence is described, the method comprising: providing at leastone gene primer pair formed by a gene forward primer and a gene reverseprimer and at least one barcode primer pair formed by a barcode forwardprimer and a barcode reverse primer, wherein the barcode forward primercomprises a first barcode at a 5′ end region and the barcode reverseprimer comprises a second barcode at a 5′ end region; combining thesample with the at least one gene primer pair and the at least onebarcode primer pair to form a mixture; and performing polynucleotideamplification reaction with the mixture, the polynucleotideamplification reaction comprising annealing between the at least onebarcode primer pair and the at least one gene primer pair and betweenthe at least one gene primer pair and the target sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of this specification, illustrate one or more embodiments of thepresent disclosure and, together with the detailed description and theexamples, serve to explain the principles and implementations of thedisclosure.

FIG. 1 shows a cross-sectional view of a series of steps in which tissuemasking selectively destroys DNA.

FIG. 2 shows a cross-sectional view of a series of steps where tissueisolation or masking is performed through lamination with aphotosensitive material.

FIG. 3 shows a top view of an embodiment in which tissue isolationtargets multiple areas of interest occurring by lamination with aphotosensitive material.

FIG. 4 shows a cross sectional view where a tissue is integrated withmicrofluidic elements.

FIG. 5A is related to FIG. 3, and illustrates a top view of a customizedchambered microfluidic device. FIG. 5B is related to FIG. 3, andillustrates a top view of a standardized chambered microfluidic device.

FIG. 6 shows a cross-sectional view of an embodiment where tissueencapsulation is performed.

FIG. 7 illustrates an embodiment in which a maskless chamberedmicrofluidic device encapsulates the tissue encapsulates a tissuewithout customized photomasking while specificity of micro-isolation isachieved through active control of arrays of valves allowing specificityof micro-isolation to be achieved through active control of arrays ofvalves.

FIG. 8 illustrates a top view of a matrix of microfluidic

FIG. 9 shows a series of steps for the parallel processing of isolatedtissue subsections.

FIG. 10 shows a series of steps for the parallel processing of isolatedtissue subsections that allows step-wise administration of biochemicalagents.

FIG. 11 illustrates an optical setup for dynamic optical array masking.

FIG. 12 provides optical microscope optical microscope images as anExample of the techniques described herein.

FIG. 13 illustrates an exemplary overlay.

FIG. 14 illustrates an exemplary overlay with barcode mapping.

FIG. 15 illustrates an exemplary device to deliver barcodes.

FIG. 16 illustrates an exemplary method on how to prepare an overlay.

FIG. 17 illustrates an assembled overlay preparation stack.

FIG. 18 illustrates filling of an overlay.

FIG. 19 illustrates assembly of an x and y overlay.

FIG. 20 illustrates an exemplary analysis device.

FIG. 21 illustrates an exemplary centrifugation and collection device.

FIG. 22 illustrates an exemplary barcode preparation.

DETAILED DESCRIPTION

Methods and systems are provided herein that allow in severalembodiments the integration of microfluidic techniques withmicro-isolation of light sensitive microscale components, such as cellsand/or tissue.

The term “microfluidic” refers to a system or device for handling,processing, ejecting and/or analyzing a fluid sample including at leastone channel having microscale dimensions. Microfluidic tissue isolationcan be customized morphologically, functionally, and a combination ofthe two.

The term “micro-isolation” refers to the isolation of micro-scalecomponents (components having a size or measure in the order ofmicrometers) and in particular of light sensitive microscale components,which includes, but is not necessarily limited to, one or morebiological components. A biological component refers to any organizedsubstance forming part of a living matter, e.g. a cell, cellularmaterial, membranes, organelles, proteins, nucleic acids and/or livingorganisms of any dimensions or a part thereof (e.g. tissues or variouscell extracts). Micro-isolation as used herein can refer to theisolation of a single nucleus, a single cell or a biological componentthereof, a group of individual cells, or a cluster of cells, or a groupof clusters of cells, or a specific region of a tissue or a portionthereof, or even cellular organelles (e.g. cell nuclei). In particular,in some embodiments, methods and system herein described allow one tosimultaneously address a distributed group of regions of interest acrossa tissue slide while each region can be a single nucleus, a single cell,a cluster of cells or a biological component thereof.

In some embodiments, the proposed integration of microfluidic techniquesand micro-isolation of cellular material molds the microfluidicarchitectures in accordance with the particular structure of eachspecific biological component to be isolated. In particular, in some ofthose embodiments, the approach described herein is mainly built aroundthe cellular material, following the tissue structure such that thedevices and methods described herein are adapted to the specificgeometry of particular tissues or other biological components. Bycontrast, in some of the traditional approaches microfluidic devices arestructured taking only in accordance with engineering considerations(e.g. path minimization, fluidic efficiency), while the biologicalcomponents in applied devices are forced to comply with theseengineering presets and are not taken into consideration.

In some embodiments, the subject matter described represents a paradigmshift in microfluidic technology because 1) microfluidic devices aredirectly integrated with, onto, or around tissue samples, in contrast tocertain conventional method of off-chip sample extraction followed bysample insertion in microfluidic devices, 2) architectural andoperational principles of microfluidic devices are mainly subordinatedto suit specific tissue structure and needs, in contrast to certainconventional method of building devices according to fluidic functionalone and without regard to tissue structure, and/or 3) sampleacquisition from tissue is to be performed on-chip and is to beintegrated with the diagnostic measurement within the same device, incontrast to the conventional method of off-chip sample prep andsubsequent insertion into a diagnostic device.

In an embodiment, a particular sample is hardwired usingphotolithographically defined masks.

FIG. 1 illustrates exemplary hardwired masks. Cells (110)inside a tissueslice (120) on a tissue support (e.g. glass slide) (130) are exposed toultraviolet (UV) light (140) through a photomask (150) comprisingblocking regions (e.g. chrome regions) (160) patterned on a transparentblocking support (e.g. glass slide) (170). The blocking regions (160)are patterned in correspondence to cells of interest (180). Theilluminating UV light (140) passes through a region of the transparentblocking support that is not blocked (175) and is prevented fromexposing an area (195) protected by the blocking region (160). DNA inexposed cells is destroyed (185) but protected DNA inside the cells ofinterest is preserved (190).

The term “illumination” refers to the exposure of light. Light can bevisible or non-visible light, and can be one or more of UV, two-photon,or multi-photon light or additional examples of light of variouswavelength suitable to be used in connection with biological componentswhich are identifiable by a skilled person upon reading of the presentdisclosure.

The term “hardwired” refers to devices, methods and systems hereindescribed or portions thereof, that are tailored for a specificbiological component of interest. For example, a particular device ishardwired if it is configured to be suitable for a specific component,e.g. a specific tissue to be investigated using methods and systemsherein described. In some embodiments, hardwired devices, methods andsystems are herein described that are tailored not only for a specificbiological component of interest, but also for a specific investigativeapproach of interest. For example, in some embodiments, hardwired masksare described that allow UV light to be directed only to areas of nointerest so that the DNA, protein, or other biological material in thoseareas is damaged to various extents and even destroyed by photodamage.In some of those embodiments, damage in areas of interest not exposed toUV is minimized to various extents and in some cases even remain intact.The term “destroy” as used in the present disclosure with reference toan item indicates a damage level able to impact at least one biologicalactivity associated to the item. The term “intact” as used in thepresent disclosure with reference to an item indicates a molecule thatpreserve all the biological activities associated to the item.

In an embodiment, cells (or another biological components or microscalecomponent) of interest can be identified, for example, using amicroscopic computerized image of the slide and appropriate customsoftware, which can convert the selection into a digital image. Thedigital mask can be fed into a direct laser writer, e.g. HeidelbergDWL66, which transfers a digital mask onto the photosensitive materialby direct writing with a resolution of 2 microns or higher (see Example1). One skilled in the art would recognize that other laser writers ormeans for transferring digital masks with even higher resolution can beused with the claimed subject matter described herein.

The term “micro-isolation apparatus” refers to a device that aids in themicro-isolation of a microscale component (e.g. a biological component,which relates to biology, life and/or living processes, such as acellular material).

The term “cellular material” refers to biological material pertaining toa biological cell. As used herein, it can refer to sub-components of abiological cell, a single intact biological cell, a group of biologicalcells, or a tissue.

The term “region of interest” as used herein pertains to a targeted areawithin cellular material. Definition of a targeted area can be of anydimensions and include one or more cellular material depending on theexperimental design of choice. For example, the region of interest canbe an area that is sought to be preserved, or an area that is sought tobe damaged or even destroyed. In a further example, the region ofinterest can be as small as a DNA molecule, or as large as an entiretissue sample, a group of topologically non-contiguous targeted areas inthe tissue sample, which are all to be isolated and/or extracted at asame or a different time.

The term “support” as used herein refers to any type of support in whichcellular material can be mounted. One type of support is a glass slide,although one skilled in the art would recognize that many materials canprovide support for cellular material.

The term “photomask” as used herein refers to the blocking supportcomprising a blocking region and a light accessible region. The term“blocking” refers to the ability of an item to hinder the passage oflight through the item. The term “light accessible” as used hereinrefers to the ability of an item to allow passage of light through theitem. In some embodiments, the photomask can be any type of transparentsupport (light accessible region) having a non-transparent region(blocking region). In some embodiments, the transparent support canfurther be at least in part, semi-transparent, or translucent and/orinclude different blocking portions with different blocking and lightaccessible capabilities (e.g. limited to one or more selectedwavelengths for one or more areas of the photomask). In someembodiments, the photomask can be a physical object (e.g. a glass slidepartially covered with chrome, or a transparency partially covered withink or other blocking material). In some embodiments, the photomask canbe purely or partially digital. For example, in some embodiments, thephotomask can comprise a series of instructions to a micro-mirror array,which operates so that some mirror elements are activated while othersare not. in some of those embodiments, they activate mirror elements toform a photomask pattern on a sample with respect to an illuminationlight reflected onto the sample by the micro-mirror array. Additionalembodiments are encompassed by the present disclosure wherein aphotomask is dynamic photomask, as the instructions are dynamicallydefined in addition or in the alternative to photomask wherein physicalblocking material (e.g. chrome coating) blocks light on a suitablesupport (e.g. glass slide).

The term “blocking region” refers to a region of a blocking support thatfunctions to block photons (e.g. non-transparent region blocking photonsby absorption). In an embodiment, a blocked region can be a region ofchrome covering the blocking support. One skilled in the art wouldappreciate that many metals or other materials could be used, forexample, to block photon exposure. For example, titanium can bedeposited on platinum, covered with a photosensitive material, and thensimilarly oxidized to produce a chemically and mechanically resistantoxide that can serve as a photon shield for agents damaging tomicro-components such as cellular material, such as UV exposure. in someembodiments, a material forming a blocking region can also further be apolarizer as will be understood by a skilled person.

The term “blocking support” as used herein refers to a support that canbe used to support a blocking region. For example, a blocking supportcan be transparent, or in general configured for allowing passage of adesired lighting in one or more areas where there is no blocking region.

Embodiments of the present disclosure can use illumination light otherthan UV. For example, a two-photon or other multi-photon approach woulduse illumination of larger wavelengths where the resulting excitationwould have an effective wavelength that is half or a smaller fraction ofthe illumination light. Such illumination wavelengths provide increasedresolution because the light intensity is a strong function of thedistance from the focal point, which allows more precise focusing of theillumination laser.

Hardwired masking can also be accomplished by other methods, forexample, methods using polarizers and/or polarizer arrays. A polarizersurface can in principle be chemically or optically modified to producecontrast between treated and untreated regions to be used as a mask forthe embodiments described herein. Some polarizers can function on theprinciple of aligned molecular structure of polymers, while others arebased on metal wire arrays. In either case, disrupting the structuralorder in chosen regions, for example by heating or melting, would makesuch regions lose their polarizer property, which would produce thedesired contrast between the regions of interest. Any hardwired methodused produces a sample where the only remaining DNA and/or otherbiological material of interest provides the sample of interest. Thus,the destroyed and/or other biological material of interest does notcontribute noise to the signal.

In an embodiment, cellular material can be micro-isolated by laminationof unwanted areas.

In an embodiment, micro-isolation by lamination provides mutualisolation of non-contiguous regions of interest, while preventing thematerial from contiguous or non-contiguous unwanted regions frominterfering with subsequent reactions. In some of these embodiments,unwanted regions e.g. DNA or proteins of interest) can be destroyed insubsequent processing of the biological component of interest. In otherembodiments, unwanted regions do not necessarily need to be damaged. Inparticular, in one embodiment DNA or other regions of interest (damagedor not) can be locked inside the laminate thus minimizing thepossibility that contamination of the region of interest can occur. Inan embodiment, a lamination approach further allows that each area ofinterest can be contacted with additional microfluidics for individualaddressability and individual extraction. In an embodiment, the natureof the photomasking (hardwired or dynamic) is orthogonal to the choiceof isolation method (e.g. DNA damaging or tissue lamination). In some ofthose embodiments, both photomasking methods are compatible with bothisolation methods.

In an embodiment, after identifying desired cells, a suitablelithography mask can be generated to protect the contents of desiredcells. In some of those embodiments, biological material such as DNA orprotein from the protected cells can be used in a number of downstreamapplications including, but not limited to, DNA sequencing, proteinanalysis, etc. The purity of such specimens will greatly enhance thevalue and information of downstream applications. In some of theseembodiments, the lamination method does not ensure destruction ofunwanted biological material, but still maximizes survival of the wantedbiological material, because the latter is protected by the blockedregions of the photomask, due to the requirements of the negativephotoresist (see e.g. FIG. 2).

In an embodiment, the lamination-based method of micro-isolation relieson the fact that DNA and many other biological materials in tissue slicesamples are neither damaged nor removed by organic solvents (e.g.ethylene, acetone, xylene). Thus, it is possible to produce a layer ofphotocurable material directly onto a tissue slice containing cellularmaterial, cure in situ by photoexposure with a desirable mask pattern,and then remove the uncured sections with organic solvents withoutdamaging the biological materials of interest. In those embodiments,photolithographic masking can thus be viewed as a way to use aphotosensitive material as a microfluidic element, which can bedynamically defined by optical methods and made to match the morphologyand analytical needs of the particular tissue sample.

FIG. 2 is an example of tissue isolation by lamination. (A) A tissue(280) containing a cell of interest (205) is fixed on a tissue support(220), (13) A photosensitive material (210) is then deposited onto thetissue (280). The photosensitive material can be deposited on thetissue, e.g. by simple application, by spinning the substance down on aspincoater, by kinetic mounting, or by using spacers (e.g. microspheresof fixed dimensions) and mechanical contact with a flat surface, (C) Aphotomask (240) comprising a blocking region (245) is then applied ontothe photosensitive material (210). (D) The tissue (280) is then exposedto UV light (230) through the photomask (240) and the photosensitivematerial (210). (E) Photoexposure through the photomask (240) produces alithographic pattern (250) inside the photosensitive material (210). (F)The photomask (240) is removed. (G) A developer is applied (not shown)to remove non-cured sections of the photosensitive material, whichleaves the areas of interest (260) open to interaction with the outsideworld. (H). The cell of interest (205) is unprotected and subjected toremoval (270) for subsequent biochemical analysis e.g. extraction orin-situ measurements) whereas unwanted cells (215) are leftinaccessible.

Those skilled in the art would see that the described process utilizes“negative” photosensitive materials (photosensitive material in whichthe protected areas are the areas that get removed), but the sametechnique can be applied with “positive” materials as well, by inversionof the mask.

The term “developer” as used herein refers to a chemical that reactswith a chemical (e.g. a photosensitive material) that has been exposedto light.

The term “lamination” or “to laminate” as used herein refers to theplacement or layering of a material, e.g. a photosensitive material,over a sample, including but not limited to, a tissue or cell sample,and the (thermal, photolithographic, or otherwise) thickening orhardening of the laminating material into a “laminate,” so that thebiological components under the laminate are locked by it and cannotcontaminate the biological components in the non-laminated areas.

The term “photosensitive material” as used herein refers to any organicsoluble or water soluble material that experiences a change insolubility in a developer solution when exposed to light, such as UVlight.

In an embodiment, one type of photosensitive material that can be usedis photoresist. One skilled in the art would recognize that manydifferent types of photoresist materials can be used such as negative(SU8) and positive (SPR and AZ) photoresists and additional photoresistidentifiable by a skilled person. One skilled in the art would furtherunderstand that other photosensitive materials, or other photocurablepolymers, can be used with the embodiments described herein. Anyphoto-curable material, which can include, but is not limited to, manytypes of polymer, elastomer, or epoxy, can be used as a negativephotosensitive material. Any substance that becomes soluble after UVexposure can act as a positive photosensitive material, for example,urethane or Polymethylmethacrilate (PMMA) and additional materialsidentifiable by a skilled person.

The use of a negative photosensitive material is shown in FIG. 2. The“dark” areas of the mask are designed or programmed to allow access tothe protected areas once the developer is applied because negativephotoresists use organic solvent developers, which neither damage norextract DNA and RNA during the development process, because the DNA andRNA are charged and hydrophilic. Subsequently, the DNA and RNA can beextracted or analyzed in situ in aqueous solutions.

The use of positive photosensitive materials causes the UV-exposedphotosensitive material to be soluble in developer, such as an aqueousalkaline developer, so that areas not protected by the mask would beopen to interaction (not shown). This allows hydrophobic molecules (e.g.hydrophobic lipid-soluble proteins) to be extracted by organic solventsthat do not damage the positive photoresist coating of the unwantedareas.

One skilled in the art would recognize that DNA and other biologicalmaterial can be damaged by UV light. Nonetheless, one skilled in the artwould also recognize that DNA most strongly absorbs light with awavelength below about 320 nm, while many photosensitive materials areactivated at higher wavelengths, for example, at 400 nm. Although somelight processing systems use a wide wavelength range, it can beappreciated that cut-off filters or other means that are commonly knowncan be used to limit the excitation light to wavelengths that are toolong to damage DNA but are short enough to expose the photoresist, whichcan include, but is not limited to, wavelengths between about 350 nm andabout 400 nm as will be understood by a skilled person.

Photoexposure can occur by any means known in the art, which includes,but is not limited to, UV exposure, light emitting diodes, or photoniccrystal devices, or alternatively or in addition, reflected onto thesample by micro-mirror arrays.

FIG. 3 shows from a top view the same process of tissue isolation bylamination as FIG. 2. (A) Clusters (310) of potential cancer cellswithin a tissue sample (320) are selected. The clusters provide aplurality of cells, each of which are targeted in a manner described inFIG. 2. (B) The selection is reflected in a photomask (either hardwiredor dynamically defined) of black spots (330). (C) A photosensitivematerial (340) is deposited onto the tissue sample (320) and a tissuesupport (not shown); the photomask (330) is aligned on top, and UV light(not shown) is directed through the photomask (330) to expose thephotosensitive material (340) over unprotected unwanted regions. (350)(D) The tissue slide (not shown) is treated (e.g. with a developer),which removes unexposed photosensitive material above the areas ofinterest. Defined access wells (370) in the photosensitive material(340) ensure that only the wanted areas can be extracted with suitablemethods (e.g. chemically) for further analysis.

The term “well” or “wells” or “access wells” or “defined access wells”as used herein describes an area around a tissue region of interest thatprovides access to that particular region of interest.

In an embodiment, the tissue lamination approach is coupled withmicrofluidic devices placed on top of the photosensitive material inorder to individually analyze regions of interest.

A photolithographically masked tissue containing cellular material canbe integrated with a microfluidic chip (e.g. consisting of a series ofchambers matching the mask) which can be fabricated in situ orseparately. The chip can be used to extract the subsamples of theselected area. The chip can also be used to supply reagents for in-situanalysis (e.g. immunoassay, PCR, RT-PCR), which allows unequaledflexibility and parallelism in the microfluidic dynamic selection oftissue areas of interest for each individual slide.

As shown in FIG. 4, access wells (410) defined in a photosensitivematerial (440) over cells of interest (430) placed on a tissue support(450) can be accessed microfluidically by producing and aligning achambered microfluidic device a microfluidic chip) (420) comprisingchannels (470) having an input (480) and an output (490).

In some embodiments, the overlay can be made of silicone, silicon, orglass.

As described herein, the term “microfluidic chip” or “chip” as usedherein refers to least one substrate having microfluidic structurescontained therein or thereon. For example in an embodiment, the chipscan be one-layer, e.g. made of silicone, where horizontal channels areconfined in a single 2-D plane, with vertical channels only forinput/output operations. The chips can also be multi-layer devices [ref.2], where each layer of the material contains its own network ofchannels. Such networks can be connected with vertical connectingchannels called “vias” [ref. 3]. The chip can contain valves, or aplurality of valves and arrays of valves. One skilled in the art wouldappreciate that any chip that can be integrated with microfluidic wellsdefined in photosensitive material to allow for highly specificextraction of desired cells can be used.

The term “channel” or “channels” or “chamber” or “chambers” as usedherein refers to a pathway formed in or through a medium that allows forthe movement of fluids, such as liquids and gases. The term “input” or“inputs” as defined herein is intended to refer to areas of themicrofluidic device where materials (e.g. proteolysis agents, gases,other liquids) can be introduced through the chambers to the regions ofinterest. The inputs further allow introduction of materials to multipleareas of interest through channels connected to the chambers.

In various embodiments, a chip herein described can provide a multitudeof analytical functions. The term “analyze” and “analytical” refer toactivities related to process of the microscale component at issue forthe purpose of detecting information related to the component. Forexample, in embodiments where the microscale component is formed bybiological material, analytical functions comprise activities directedto process the biological material to identify information concerningand/or originating from the material which are identifiable by a skilledperson. For example, in an embodiment, the integrated chip can uptakemicro-isolated cells, lyse such cells, capture DNA, RNA, and/or otherbiological materials by specific or general hybridization assays tomagnetic nanoparticles, which can then be extracted from the chip fortraditional PCR analysis off-chip. In an embodiment, the chip can alsouptake the micro-isolated cells, lyse such cells, and perform PCRon-chip. In some of those embodiments, on-chip PCR allows subsequentdetection of specific genes or mutations by molecular beacons or on-chipspecific hybridization arrays. In an embodiment, the chip can alsouptake a biological components such as micro-isolated cells, lyse suchcells, and then perform immunoassays on intracellular proteins, e.g. forproteomics-level expression analysis. In an embodiment, the chip canalso be used to extract the cell regions micro-isolated by thelamination techniques described herein, separate the clusters intosingle cells, then feed each cell into a single-cell analysis device,e.g. for DNA sequencing, mutation detection, or gene expressionanalysis. Such devices as the enumerated examples can be used infundamental research, and can also be adapted as biomedical diagnostictools, e.g. in oncology and pathology. Additional variations of thesedevices, wherein same or further functionalities are added or combinedare encompassed by the present disclosure as would be understood by askilled person.

The term ‘output” or “outputs” as defined herein is intended to refer toareas where the cellular material from the regions of interest can beextracted for further analysis. Input/output operations can be conductedby microfluidics, thus preventing contamination, waiting for diffusion,and excessive dilution of the sample. Also, the chip itself can haveanalytical function, e.g. microfluidic immunoassays or microfluidic PCRand RT-PCR.

In some embodiments, the described integration of tissue laminationmicro-isolation with microfluidic devices allows the leveraging of theadvantages of microfluidic handling (e.g. micro scale, fast diffusion,parallelism, integrated mechanical and/or chemical functionalities) withthe advantages of the described micro-isolation technique (e.g. highspecificity, parallelism, low cost, flexibility, and/or dynamicmasking). In some of those embodiments, this architecture providesflexible sample-specific interface between the unique patterns of theparticular sample morphology and the hardwired architectures ofconventional microfluidic devices.

FIG. 5A shows a top view demonstrating a hardwired multi-chamberedapproach wherein regions of interest (from FIG. 3) are connected viachannels. A laminated tissue sample (550) is integrated with a chamberedmicrofluidic device (as shown in FIG. 4), whose channels (510) connectto chambers (as described in FIG. 4) over the laminated tissue'scellular areas of interest (540). An input (520) allows introduction ofcomponents necessary to collect extracts into a single output (530).FIG. 5A shows a single input and output, although multiple inputs andoutputs are possible. In this particular approach, the entire chip iscustomized to the extraction needs of the particular sample, although asdescribed herein, multiple approaches are possible.

In an embodiment, the lamination is customized to the particular sample,but the rest of the microfluidic device can be standardized and thusused universally with any and all tissue samples. As used herein, auniversal microfluidic device is a device that can be standardized andused with multiple tissue samples that have been selectively ornon-selectively isolated. For example FIG. 5B shows a top viewdemonstrating a standardized multi-chambered approach. In this approach,a laminated tissue (550) is integrated with a chambered microfluidicdevice (as shown in FIG. 4), whose standardized matrix of channels (560)connect to chambers (as described in FIG. 4) over the laminated tissue'scellular areas of interest (540). An input (520) allows introduction ofcomponents necessary to collect extracts into a single output (530).FIG. 5B shows a single input and output, although multiple inputs andoutputs are possible. Those skilled in the art would appreciated that astandardized multi-chambered chip can be configured in one of manypossible ways in order to allow microfluidic channels to cover a tissuesurface adequately in order to extract desired cells from randomlydistributed locations. For example, the grid can be rectangular as shownin FIG. 5B, or a binary tree of parallel channels. In addition, auniversal extractor device can be combined with a sample-specificmicro-isolation technique. In embodiments, wherein the spacing in a gridor mesh of channels is not bigger than the expected size of wantedclusters, a fluidic connection between one or more wanted cluster withat least one of the extraction channels can be performed. Thus in someof those embodiments a universal extractor device can be combined with asample-specific micro-isolation technique (e.g. the described laminationmethod). In some embodiments, in particular the described combinationcan have the benefits of both techniques in the same system—low cost anduniversal applicability offered by the standardized chip with the highspecificity and sample-specific customization of the micro-isolationtechnique.

In an embodiment, a tissue is completely encapsulated inside amicrofluidic device, to allow for full surface access.

Tissue encapsulation captures a tissue of interest between two separatemicrofluidic devices, which allow simultaneous access to two surfaces.If the slice is sufficiently thin, fluidic communication is ensuredthrough the slice. Such communication allows more efficient and reliableextraction of desired samples, as a resuspension liquid can be used topush desired material out of the tissue matrix. This approach allowsextraction of desired material by microfluidic/hydraulic means withoutthe need for more aggressive chemical treatments.

FIG. 6 illustrates an embodiment where complete encapsulation is shown.A tissue slice (630) covered in a photosensitive material both above(620) and below (625) the tissue slice and defined by access wells bothabove (660) and below (665) the tissue slice that can be integrated withmicrofluidic devices (e.g. microfluidic chips) positioned above (610)and below (615) the cells of interest and having chambers from bothabove (670) and below (680) the tissue slice. This architecture allows amore efficient input (640) and output (650), while an area of contact(690) can be doubled for better access to the cells of interest (695).

In an embodiment, tissue encapsulation allows the use of 3Dpolymerization for in-situ chip construction around the 3D tissuesample. This can be done, e.g. by using direct laser writing and 3Drastering to build the desired architectures such that the monomermaterial for the chip can be spread thick over the tissue sample. Thelaser can then polymerize the chip material in the desired shape overthe tissue sample. The tissue is completely submerged in a monomer,while a 3D chip is built around it, thus allowing microfluidic access tothe sample from all directions. In some embodiments, performed accordingto this approach, the photocurable polymer of the chip material itselfcan be used as a photosensitive material, wherein a tissue slice can beplaced inside the polymer prior to 3-D photopatterning (e.g. before a3-D) photopatterning commences)

In an embodiment, multiple areas of interest are addressed withindividual channels without masking.

Individually and collectively controlled arrays of microvalves, whichallow the same architecture to address a customizable subset of chambersof access loci within the matrix, can provide the ability to matchparticular regions of interest on the particular tissue sample. A chipwith a dense pore matrix allows the differential opening of particularpores for a short time such that only material confluent with the porewould flow from the sample into the chip for analysis without masking.

Maskless microfluidic encapsulation is shown in FIG. 7. A tissue slice(710) is encapsulated in a dense pore matrix microfluidic device bothabove (745) and below (750) the tissue. Individually addressable valvesthat are closed are shown marked with an “X” (770) as opposed to valvesthat are open (760) to allow flow through the open channel (755). Theopen valve (760) forces a pressure drop that ensures input flow (720)and forces material from a cell (740) through a pore (730) into anoutput channel (780) and through an output (790) for analysis.

An embodiment provides tissue micro-isolation by microfluidic matricesfor parallel analysis of subsamples with preserved morphologicalcontext.

According to an embodiment, microfluidic matrices with highly parallelsingle-cell analysis is based on the combination of a nanofabricatedmicrofluidic matrix and a tissue section. This allows a matrix ofmillions of microfluidic wells to be filled with biochemical reagentsand contacted to a tissue section deposited on a support. For example,each well can contain Proteinase K and sequencing reagents. TheProteinase K digests the tissue and releases the contents of each cellinto its adjoining well, where they mix with the PCR reagents bydiffusion. The entire system is contacted to a thermally controlledaluminum plate, to perform standard or isothermal PCR. Mutant genes canbe amplified by appropriate selection of primers and reported byfluorescent probes. Signal detection is done by scanning the entireslide on a fluorescence scanner or by fluorescence microscopy performedone sector at a time followed by digital assembly. The result is ahighly parallelized single-cell (genetic) analysis of the entire tissue.

An exemplary matrix of microfluidic wells according to an embodimentherein described is illustrated in FIG. 8, which shows a top view ofaccess wells (810) inside a matrix (820) (built in e.g. glass silicon,or silicon-on-insulator). Access wells (810) can be defined, forexample, by spreading a photoreactive material on a substrate, exposingthe photoreactive material to UV light through a photomask, developingthe photoreactive material, and etching the exposed areas. Followingremoval of the photoreactive material, the result is defined accesswells in the substrate with the same geometry as the photomask. The term“dense-pore matrix” as used herein refers to a matrix having densepores.

FIG. 9 shows an exemplary illustration of how a matrix of microfluidicwells can provide access to individual cell nuclei for independentreactions. Microfluidic access wells (910) are defined in a substrate(920) (e.g. glass or silicon). Next, the access wells (910) are filledwith a fluid mixture (930) containing digestion and reaction agents(e.g. Polymerase Chain Reaction (PCR) reagents, and fluorescent probes).Next, evaporation is performed to uniformly decrease the fluid mixturelevel (940), leaving space at the top (945) of the microfluidic accesswells. Next, a tissue support (950) is aligned on top with a tissueslice (960) facing downward. Next, an assembled construct (955) isclamped together and vertically turned over, allowing the fluid mixtureto flow by gravity and cover (975) the tissue. Next, the digestionagents contained in the fluid mixture break down the tissue (965),releasing the cellular contents into the microfluidic access wells(910). The reaction reagents within the fluid mixture complete thereactions and fluorescent probes (980) reveal results.

In an embodiment, releasing the cellular content of the tissue can beperformed by digestion of the tissue performed using Proteinase K. orother techniques of chemical digestion such as the ones described in[ref. 4,5] which allow multiple analysis of different molecules in thecellular component. For example, in an embodiment techniques can be usedthat allow immunoassay analysis of the extracts as well as DNA andprotein analysis at various scales as it will be understandable by askilled person.

In several embodiments, the nanomatrix technique herein described can bemodified in view of the specific reagents, biological component, desiredresult and experimental design as will be understood by a skilledperson. For example, in an embodiment, a matrix of microfluidic wellscan provide access to individual cell nuclei where a two-step processallows separate biochemical reactions to occur. By way of example, FIG.10 shows a process in which cells can be digested with Proteinase Kprior to a PCR reaction. In the illustration of FIG. 10 tissue (1030) isplaced atop of a support (1010) and a photosensitive material (e.g.negative photoresist) (1020) covers the tissue (Panel A). Next, aphotomask (1050) having—blocking regions (1055) is placed over thephotosensitive material and exposed to UV light (1040) (Panel B). Next,the photomask is removed and an organic solvent developer removes thephotosensitive material from the unprotected areas, leaving definedaccess wells (1060) (Panel C). The defined access wells are filled withsolution containing Proteinase K (1065) (Panel D), which digests exposedtissue (1070) and releases the DNA into the solution in each well (PanelE). Heating deactivates the Proteinase K and lyophilizes DNA in place ineach respective well (1075) (Panel F). Next, a corresponding matrix ofwells is etched in a well support (e.g. silicon, glass, orsilicon-on-insulator) (1080), which is filled a solution containing PCRreagents (1085) (Panel G). The assembly is then mechanically securedtogether (e.g. clamped) providing water-tightness between compartments,and then turned over, allowing the PCR solution to resuspend thelyophilate within each well (1090) (Panel H). PCR can proceedsimultaneously yet separately, in which fluorescent probes (1095) revealresults of the reaction (see starbursts Panel I). Data acquisition canbe performed e.g. on a fluorescence scanner or by an opticalfluorescence microscope, where the wells are optically accessed by theside of the glass slide in panel I.

In the embodiment exemplified in FIG. 10, when the surface density andsize of the wells are correctly chosen, most wells will adjoin one andonly one cell. In some embodiments, wherein the biological component isformed by cells, an expected optimal well size is about the same as thesize of a mammalian cell (˜20 μm). However, in those embodiments, oneskilled in the art would recognize that the size and spacing of thewells can be optimized to ensure that the overwhelming majority of wellscontain just one cell. This would maximize purity of the sample in eachwell, and thus maximize specificity and reduce noise in an analyticaldetermination.

In an embodiment, a maskless microisolation apparatus methods andsystems are described that are configured according to the microscalecomponent of interest and experimental design and do not require(although they could include) photomasks and/or photosensitive material.For example, in some of those embodiments, a maskless microisolationapparatus can have randomly placed access wells that are configuredrelative to the biological material of interest and the experimentaldesign. In particular, in embodiments where the biological material iscomprises cells and the experimental design is directed to isolateand/or analyze individual cells, a dense porous “honeycomb” arrangementof predetermined access wells can be used in a device, methods orsystems herein described such that the pre-existing access wells can beplaced accordingly over cellular regions according to the specificanalysis of choice (e.g. to perform protein and/or DNA analysis ofregions of interest selected).

In some embodiments, the configuration of a matrix of access wellsherein described is not limited to desirable areas alone. In some ofthose embodiments, some or all wells can be analyzed simultaneously butseparately, so that no predetermined regions are necessary. Accordingly,in an embodiment, of devices methods and systems herein described aphotomask can be designed to single out only the areas of interestand/or to include a repeating regular or irregular geometric pattern ofchoice (e.g. circles, squares, or hexagons in rectangular, checkered, orhoneycomb formation) of appropriately chosen size and spacing, e.g. tocontain only one microscale component or portion thereof (e.g. one cellper well).

In several embodiments, wells of a masked or maskless matrix of accesscan include one or more reaction mixtures. A reaction mixture can be anymixture containing components necessary for a biochemical reaction tooccur. Reaction mixtures can include, but are not limited to, componentsnecessary for PCR, real-time PCR, RT-PCR, flow cytometry, fluorescentlabeling, FRET, DNA sequencing, protein-protein interaction assays,immunoassays, protein-nucleic acid assays, and any other biologicalreaction known in the art.

A matrix of microfluidic wells allows incomparable parallelism inextracting the sequencing information while preserving the morphologicaland contextual information from the tissue sample. It enableslarge-scale mapping of two-dimensional spatial distribution of mutationsacross a tumor section. If such maps are made of consecutive sections ofthe same tumor, a three-dimensional distribution of mutations within atumor can be digitally assembled. Furthermore, such 3D maps can begenerated for analogous tumors in multiple test animals at differenttemporal points of tumor evolution. Thus, the temporal evolution of a 3Ddistribution of mutations can be assembled.

In many embodiments, the optimal well size is the same size as amammalian cell (approximately 20 μm), although one skilled in the artwill recognize that different well sizes can be used for differentapplications.

In an embodiment, an active array of masking material replaces aphysical mask with micro-mirror arrays. As illustrated in FIG. 11, whichis a dynamic process allows the targeted positioning of tissueselection. First, cells and/or tissue (1160) are shown to an operatorand a camera (1110) takes an image of cells and/or tissue through anadjustable mirror (1120), a Digital Light Processing (DLP) mirror (1130)and a photosensitive material (1140). The image shows the cells and/ortissue (1160) placed on a tissue support (1150). Upon observation of theimage, an area of interest is selected by programmable patterning theDLP mirror (1130). Second, the adjustable mirror (1120) is adapted to bepositioned accordingly so that UV light from a lamp (1170) can bedirected through a long-pass filter (1180) and through the programmedDLP mirror (1130) onto the photosensitive material (1140) and directedto destroy the DNA of the cells in the region of interest (1160).

A long-pass filter indicates a device that operates to allow all lightcoming from the UV light having a wavelength above a certain value, e.g.about 350 nm. Long-pass filters are standard optical elements known topeople skilled in the art. The particular long-pass filter suitable forthe embodiment exemplified in FIG. 11 is configured to ensure thatvirtually all light coming from the illumination source has a wavelengthabove the cut-off value of about 350 nm. The usual structure of longpass filters is a Bragg stack of layers of dielectric materials withcarefully controlled thicknesses. The thickness and refractive index ofeach layer sets up destructive interference for a narrow band ofwavelengths that are meant to be stopped. Making a stack of such layersensures that a wider cumulative range of wavelengths is stopped by thefilter. In this particular case, the cut-off value is 350 nm, becausewavelengths above it are too long to damage DNA when DNA is chosen asmicroscale component of interest, but short enough to expose thephotoresist correctly. In the exemplary system of FIG. 11 the long passfilter is for the tissue lamination method, which necessitates theexposure of the photoresist which becomes the laminate. People skilledin the art (e.g. optics and engineering) understand all the possiblevariations of long-pass filters in devices, methods and filters hereindescribed.

The term “digital image” refers to an image generated by a computer orother suitable electronic device. In an embodiment, a digital image canbe provided, for example, by a set of instructions in software on acomputer controlling the optical hardware. In an embodiment, the imagecan be a 2-D image. In an embodiment, the digital image can also be 3-D,e.g. in embodiments, when a device is provided for tissue encapsulationby 3-D rastering of the photocuring illumination, as described herein. A“digital mask” refers to the masking of a region of interest of cellularmaterial based on a digital image as opposed to a physical mask, e.g. achrome mask.

In an embodiment, tissues can be micro-isolated without the need for aphysical mask for UV shielding. Instead, a UV laser, e.g. HeidelbergDWL66®, can be focused directly onto the necessary spots in thephotoresist on top of the tissue for lamination or in the tissue itselffor destruction of biological material in the tissue such as DNA. Theresolution can be 2 microns or better, and the desired cells can beskipped in the rastering process. Different laser heads can be used forthe different regions of the slide. For example, appropriate softwarecan guide the laser with a 2-micron head around the immediate vicinityof the cells of interest, while the rest of the slide area is exposed bybroader strokes, e.g. with a 30-micron head.

In an embodiment, active masking arrays utilize LCDs (liquid crystaldisplays). An illumination light would be polarized along one axis,while the LCD elements would be polarized along one axis to disallow andanother axis to allow the passage of the UV light. The cells of interestare protected by having the corresponding elements in the array beperpendicularly aligned, while the unwanted cells would have theirelements aligned in parallel with incident UV illumination.

In an embodiment, dynamic masking using fiber optics can be produced byarrays of LEDs (light emitting diodes). This approach allows theutilization of increasing smaller wavelengths as current technologybuilds LEDs at smaller wavelengths. Individually addressable elementscan be built at the microscale, producing macro-sized arrays ofthousands or millions of individually addressable LED elements. Suchindividually addressable LED elements allow respective areas on thephotosensitive material to be individually photopolymerized to providethe tissue lamination methods described herein.

In an embodiment, fiber optics is used in a way similar to intensifiedCCD cameras. Bundles of fiber optic cables are arranged to produce anactive array of illuminators. This bundle can be coupled to an LCD arrayat the input of illumination light, while the output is coupled to thetissue slide. Then the output size of each fiber can be made smallerthan the input size, producing both light transduction and sizereduction.

In an embodiment, active masking array uses photonic circuitry to definedynamic optical arrays. A photonic circuit can in principle be built togenerate an array of individually addressable optical outputs. Whenpositioned over a tissue slide, the individual addressability of opticaloutputs provides the capability for individual UV exposure of tissueareas that are chosen to be discarded.

In an embodiment, active masking array uses photonic circuitry to definedynamic optical arrays. A photonic circuit can in principle be built togenerate an array of individually addressable optical outputs. Whenpositioned over a tissue slide, the individual addressability of opticaloutputs provides the capability for individual UV exposure of tissueareas that are chosen to be discarded.

In an embodiment, active masking array uses micro- and nano-lasers fordynamic arrays. These lasers can be fabricated in arrays, where eachlaser is still individually addressable. Software and electrical outputscontrol which laser is active, e.g. by electrical pumping or electricalcontrol of polarization shielding against pumping illumination.Microfluidic devices can further follow a combination of morphologicaland functional customization. For example, in the particular techniqueof multi-layer elastomer microfluidics, the elastomeric layer thatcontacts the sample can have a photolithographically defined morphologythat matches the regions of interest in the tissue sample, while otherlayers can follow a matrix or array structure built for functionalprogrammability As an example, see FIG. 5A, where a uniform matrix ofchannels overlays the wells of the regions of interest, ensuringextraction. Thus the extraction matrix can be standardized and thusproduced inexpensively, while the laminating layer is kept specific tothe particular tissue sample. One skilled in the art would appreciatethat such a combination is clearly not limited to extraction alone,because a device of any processing or analytical function can beintegrated with a sample-specific micro-isolation stage. In someembodiments, the specific functionality or purpose of the device can becombined with the high specificity and sample-specific customizationoffered by the described micro-isolation techniques. In some of thoseembodiments, this approach provides a low cost of standardization with ahigh specificity of sample-specific extraction

In some embodiments, the methods and devices described herein overcomevarious problems e.g. by providing a general microfluidic bottoms-upsample-specific customization method. Such a method naturally leads torapid, parallelized, and highly specific micro-isolation of the desiredcell subpopulation (e.g. cancer cells from a tumor) directly from tissuesamples. In particular, in some embodiments, devices, methods andsystems herein described preserve the structural integrity of most ofthe tissue, thus preserving its inherent morphological information. Insome embodiments, a PCR nanomatrix technique is described that allowsparallelized large-scale high-throughput genomic mappings across thetissue sample.

In several embodiments, methods herein described allow convenientapplication in a number of methods to separate normal from cancer cells(See Ref. 1). Methods herein described are not necessarily dependent onuse of fresh tissues, and are applicable to most human cancer specimens,which are usually fixed in formalin and paraffin-embedded. In someembodiments, devices methods and systems herein described allow toprocess wanted cells (e.g. cancer cells) minimizing the background noiseof unwanted cells (e.g. non cancerous cells). In some of thoseembodiments, devices, methods and systems herein described allow a lessexpensive and less labor-intensive of certain methods of the art where atrained operator must manually identify and then individually addresseach cell to be analyzed using an expensive and complex laser microscopysystem.

In some embodiments, devices, methods and systems herein described canbe performed in microfluidics. In particular, in certain embodiments,microfluidics is the micro-manipulation of fluids, and can be integratedwith biochemical applications for microscale analyses of cells withfurther implementation identifiable by a skilled person.

In several embodiments, devices methods and systems herein described areconfigured to combine sample specific microisolation with standardizedprocessing and/or analysis chip. Microisolation can be used to controland in particular increases up to maximize specificity of selection. Useof a standardized processing and/or analysis chip can provide aneconomic and industry friendly way to materialize the technique inpractice.

According to several embodiments, microisolation devices methods andsystems are described wherein: cellular material or other microscalecomponent is positioned on a substrate; a sample specific microisolation method herein described is applied to the cellular material orother component to obtain a substrate presenting the cellular material,a microfluidic universal device is connected with or contacted to thesubstrate presenting the cellular material, the universal device beinganalysis specific and sample non specific; and a microisolation methodsis used to extract the material from the desired regions on theparticular sample and the universal device is used to collect and/oranalyze the extracted sample

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice for testing of theproducts, methods and system of the present disclosure, exemplaryappropriate materials and methods are described herein as examples.

EXAMPLES

The devices, methods and systems herein described are furtherillustrated in the following examples, which are provided by way ofillustration and are not intended to be limiting.

In particular, the following examples illustrate exemplary hardwired andlaminated devices and related methods and systems. A person skilled inthe art will appreciate the applicability and the necessarymodifications to adapt the features described in detail in the presentsection, to additional solutions, devices, arrangements, methods andsystems according to embodiments of the present disclosure.

Example 1 Hardwired Masking Using Photolithographically Defined ChromeMasks

Cells of interest are identified using a microscopic computerized imageof the tissue slide and appropriate custom software, which converts theselection into a digital image. The digital mask is fed into a directlaser writer, the Heidelberg DWL66®, which transfers a digital mask ontothe “positive” photosensitive material deposited on top of achrome-covered plate, by direct writing with a resolution of 2 microns.The plate is then developed to remove the exposed photoresist, whichleaves the exposed areas susceptible to chemical etching. The etchingremoves the unprotected chrome, and the rest of the photosensitivematerial is removed, e.g. by overdevelopment or exposure to a stronglyalkaline solution. The remaining chrome pattern is quickly oxidized byatmospheric exposure, typically within 30 sec, which produces a chromemask specific to the particular tissue sample. An exemplary hardwiredmasking using photolithographically defined chrome masks is illustratedin FIG. 1.

Example 2 Application of Tissue Lamination Approach to Adrenal GlandTissue Slides

The tissue lamination technique was applied to adrenal gland tissueslides prepared by routine clinical methods. Photoresist SUS-2005 wasdeposited onto a tissue by spinning the slide on a WS-400B-6NNP/LITEspincoater. The slide was pre-baked at 65° C. Next, the slide wasexposed to UV filtered with a 368-nm high-pass filter at an MA-6 maskaligner, through a chrome-on-glass mask bearing the pattern of a USAF1951 resolution chart. The chart was chosen as a mask to provide aneasily identifiable reference in terms of size of the defined featuresin photoresist on top of the tissue.

The slide was then post-exposure baked at 95° C. and developed in SU8developer, which contains organic solvents. Finally, each slide wascharacterized on a profilometer (Alpha-Step 500) to measure the heightof the fabricated features. Tissue slice thickness was measured up to 5um tissue, while the photoresist layer was ˜7 μm high. FIG. 12 shows the“windows” defined in the photoresist. The dimension defined on thetissue was ˜12 μm width, which is smaller than a typical mammalian cell(20 μm).

The tissue section is essentially unchanged after photolithography (seeFIG. 12A), except for the discoloration of unmasked areas due to theleeching of the hematoxylin and eosin staining by the organic solvent ofthe photoresist developer. Some of this discoloration extends under themask (see FIG. 12A, 12D), likely because the organic solvent is a verysmall molecule that can penetrate through the tissue to reach the maskedareas. An alternative explanation is that the dye can diffuse out intothe wells during the digestion and extraction process, leaving the areasof immediate proximity to the wells. It is noted however that the nucleiremain in the unwanted areas but are extracted from the wantedareas—therefore, the unwanted DNA cannot diffuse out the way the dyecan.

The laminated areas of the tissue appear far brighter than the exposedtissue (see FIG. 12A) because the refractive index of the photoresistmatches the refractive index of the tissue better than air, while thephotoresist also mechanically smoothens the surface roughness of thetissue. Thus surface light scattering and refractive divergence aresignificantly reduced, and the intensity of the detected light isincreased over the laminated areas, in comparison to non-laminatedtissue.

To extract the exposed tissue, a drop of extractions solution (10 mMTris-HCl, 2 mM EDTA, pH 8.0, with 10 mg/ml Proteinase K) is placed ontop of the masked slide and incubated at 56° C. in a humidity chamber.The Proteinase K digests the tissue, releasing the DNA into solution,which is then suitable for amplification by PCR. The slide afterdigestion (FIGS. 12B, 12C, 12D) shows the removal, with sharp boundariesdefined by the mask, because Proteinase K is a large protein and thusunable to diffuse through the tissue. As seen in FIG. 12B, the digestionis less efficient with smaller features, because the photoresist ishydrophobic and so surface tension works as counter pressure against theentry of the extraction solution into the smaller holes.

The follow sections will describe additional embodiments of methods anddevices.

In some embodiments, as visible in FIG. 13, an overlay (1305) maycomprise a plurality of openings (1306) that allow access to a tissuesample on a support. In some embodiments, the overlay may comprise aplurality of wells (106) that allow access to the tissue and can alsoallow filling of the wells with reagents. For example, the overlay(1305) may have a specific thickness to allow the wells (1306) tocontain a PCR reagent. When the overlay (1305) is contacted to a tissue(1307), the reagents in the wells are in contact with the tissue (1307),either automatically or by an action performed by a user or machine,such as a pumping action that applies pressure to the reagents in thewells to enable contact with the tissue.

The tissue (1306) may be on a support (1309), such as, for example, aglass support such as a glass slide. A side view of the structure isalso visible in FIG. 13, where the overlay (1315) comprises a pluralityof access wells (1320). The tissue layer (1330) is between the overlay(1315) and the support (1325). A sealing layer, or seal, (1310) may bepositioned on the overlay (1315), for example to prevent the PCRreagents from being contaminated from above.

In some embodiments, an overlay can comprise thousands of openings (forexample, 5×5 μm to 1000×1000 μm) which are pre-loaded with reagentsenabling multiplex PCR reactions for next generation sequencing (NGS)library generation and subsequent sequencing (and other types ofbiological reactions). The overlay may have openings with uniform size,or may have different sizes in different areas of the overlay. Theoverlay can be placed over a tissue slide. An example of tissue slidesare the formalin-fixed, paraffin-embedded (FFPE) tissue slides. The FFPEslides can be hematoxylin and eosin stain (H&E) stained. The overlay canbe affixed to the tissue creating thousands of individual “reactionwells”, and a seal can be placed on top of the overlay to contain thewells and the biological reactions (PCR and other types of reactions).Reactions occur on/with single cells or small clusters of cellsdepending upon the size of the overlay's openings. Therefore, the sizeof the openings can be chosen according to the desired application.

In some embodiments, each reaction well is pre-loaded with the samePCR/NGS reagents and a “well-ID” barcode sequence tag. The barcode tagidentifies the specific well and enables a unique measurement of eachwell. Each measurement can be identified by its attached barcode,enabling several applications. For example, the same gene with differentmutations can be identified in different regions of the tissue slide.Alternatively, different genes may be identified in different regions,or a combination of different genes and mutations can be identified. Thespatial locations of the genes, identified by each unique well barcode,enable a two-dimensional mapping of the tissue slide.

A different pair of barcodes is located at each well and is used toidentify the location of the reaction for subsequent analysis. Columnsand rows are filled with different barcodes or “well-IDs”. For example,referring to FIG. 14, a first barcode A can be filled in all the wellsof a first horizontal row (1405), with a second barcode B in a secondrow (1410) and subsequent barcodes C, D . . . in subsequent rows. Forexample, the rows can be referred to as an x axis. The columns, or yaxis, are also filled with unique barcodes, different from the barcodesof the x axis. For example, in a first column (1415) a barcode 1 can beintroduced into all the wells of the first column (1415), with a secondbarcode 2 in a second column (1420), and subsequent barcodes insubsequent column. The barcodes and columns can be also termed ashorizontal (rows) and vertical (columns), even though both rows andcolumns are in the plane x,y, the plane of the tissue slide. The rowsand columns can also be termed as lines, with rows being perpendicularlines, relative to the column lines.

In some embodiments, the assay procedure comprises multiple steps asfollows. In a first step, the H&E stained tissue slide is imaged. Insome embodiments, the whole slide is imaged through whole slide imaging(WSI).

In a second step, the pre-loaded overlay is placed on-top of the FFPEhematoxylin and eosin stain tissue slide and a seal are placed on top ofthe overlay. The overlay contains reagents for the desired biologicalreaction—for example, PCR reaction reagents for NGS library generation,which is then sequenced.

In some embodiments, the assay procedure comprises multiple steps asfollows.

In a first step, the H&E stained tissue slide is imaged. In someembodiments, the whole slide is imaged through whole slide imaging(WSI). In a second step, the pre-loaded overlay is placed on-top of theFFPE hematoxylin and eosin stain tissue slide and a seal are placed ontop of the overlay. The overlay contains reagents for the desiredbiological reaction—for example, PCR reaction reagents for NGS librarygeneration, which is then sequenced.

Subsequently, the slide/overlay/seal combination is placed in a thermalcycler to carry-out the desired biological reactions in the individualwells made by placing the overlay on the tissue slide (and seal on theoverlay). A thermal cycler, and understood by the person of ordinaryskill in the art, is a device that cycles the temperature, for exampleas required by PCR protocols.

Upon completion of the thermal cycling, the slide/overlay/sealcombination can be imaged again. In different embodiments, imaging canbe carried out at the same time of the thermal cycling, or at differenttimes, for example at the beginning, and after each cycle, or only atthe end of the entirety of cycles. Imaging can be used to check whetherthe PCR protocol was successful, and whether each thermal cycle isincreasing the amount of DNA. Accordingly, the imaging can be used as atool to verify the positive outcome or positive development of thereactions.

The slide/overlay/seal combination is removed from the thermal cyclerand placed into a centrifuge and centrifuged in order to collect thelibrary generated in the biological reaction. The NGS library can thenbe prepared for sequencing. In some embodiments, optional PCR, clean-up,and sequencer specific preparation may take place at this time as well.

Subsequently, morphogenomic analysis can be carried out—image data iscombined with sequencing data and analyzed by a software that maps thesequenced data to the tumor section and cell types.

As visible in FIG. 14, a different pair of barcodes is loaded in eachwell. To realize this combination, columns and rows are filled withdifferent barcodes. A microfluidic device can accept the differentbarcoded reagents and distribute them to column or rows. In someembodiments, the device can fill both columns and rows, while in otherembodiments, one device is used to fill the columns and one device tofill the rows. For example, in the embodiment wherein the number ofcolumns is equal to the number of rows, the same device can be used forthe columns and rows, simply by rotating it by ninety degrees.

FIG. 15 illustrates an embodiment of a distributor chip that can be usedto fill the wells of an overlay. For example, one input (1510) canbranch out in multiple channels (1505), for example with a binary treesplit. The microfluidic channels branch out until there is one channelfor each of the overlay wells in a single row (or column). For example,direction (1520) can be termed the y direction, and direction (1525) canbe termed the x direction. Input (1510) is able to fill each overlaywell at x=0, along the y axis. Additional microfluidics channels andinputs can be placed at each successive x position (1515), along the xaxis. Each of these additional microfluidic channels can fill all theoverlay wells along the y axis, at each subsequent x position. Theseadditional structures are not shown in FIG. 15 to avoid cluttering theview, but can be understood by the person of ordinary skill in the art.One input (1530) and binary tree structure at the last x position isillustrated in FIG. 15, as an example. Similarly, the tree channelstructures can be rotated by ninety degrees, or the chip itself can berotated by ninety degrees around a vertical axis, to allow each input todistribute identical reagents to all wells in the same corresponding rowalong the x axis along a corresponding y position.

The person of ordinary skill in the art will understand that similar,but modified, microfluidic structures may be used, having modifiedbranching or inputs depending on the application. It may also bepossible, in some embodiments, to have duplicate barcodes at differentinputs, instead of a unique barcode at each input.

The device of FIG. 15, or a similar device, can then be rotated ninetydegrees to apply other barcodes, so that the end result is a uniquecombination of barcodes at each overlay well. For example, in a simpleexample with four overlay wells, four unique barcodes A, B, 1, 2, areneeded, so that the end result would map the four wells with A1, B1, A2,B2. For an example with 25 overlay wells, a total of 10 unique barcodesis needed to have a unique two-dimensional mapping of the 25 wells. Insome embodiments, the number of rows and columns is different. Ingeneral, a×b overlay wells can be uniquely mapped with a+b uniquebarcodes. Therefore, the embodiment with each unique barcode and eachinput allows a completely unique mapping code at each well in theoverlay, thus creating a two-dimensional barcode map.

FIG. 16 illustrates an exemplary method on how to prepare an overlay. Adistributor chip as described in FIG. 15 is illustrated in more detailsin FIG. 16. Several layers are identified, serving different purposes.In the example of FIG. 16, the binary tree splits only to a total offour overlay wells, for clarity of exposition. A first layer, a toplayer, is illustrated (1605). Layer (1605) comprises a binarymicrofluidic tree (1610), and holes for alignment rods (1615). The holesfor the alignment rods continue throughout subsequent layers and allowthe insertion of alignment rods (1620). These rods allow the alignmentof all layers, so that the layers can be assembled in the correctposition, therefore avoiding wobbling and misalignment of the layers. Insome embodiments, the rods can be screwed onto the layers and a supportat the bottom, to allow compression of the layers during filling of thechannels with the reagents. Layer (1610) can be made of differentmaterials, for example polydimethylsiloxane (PDMS) or 3D printedphotocurable resins. The channels within the layer can be fabricated byphotolithography, etching, 3D printing, or other fabrication techniques.

A next layer (1625), which can be termed the overlay layer, can be made,for example, of silicone. In some embodiments, layer (1625) can be madeof silicone-based pressure sensitive adhesive, or silicone coated. withadhesive on one or both sides, to allow attachment to other layers orsupports. The overlay layer (1625) can comprise, for example, fivesub-layers: a plastic foil, e.g. pvc, on top of an adhesive deposited onthe top surface of a microfluidic matrix (e.g. made of silicone),followed by another adhesive deposited on the bottom surface of themicrofluidic matrix, and finally another foil at the very bottom. Thewells of the matrix are defined in all sublayers of the overlay, e.g. bylaser cutting them all the way through the overlay. At a later stage,the plastic foils can be removed to allow attachment of the overlay toother layers, supports, tissue, or seals. The next layer (1630) can betermed the receiver chip. The receiver chip (1630) comprisesmicrofluidics channels that match those of the overlay (1625). Thechannels in chip (1630) can be fabricated, for example, by 3D printingof photocurable resin. The stopper (1635) can be made, for example, ofPDMS. An additional layer (1640) has matching microfluidic channel whenattached to the stopper, the channels corresponding to the receiverchip. The layer (1640) can be fabricated by 3D printing, e.g. as a copyof the receiver chip.

The layers of FIG. 16 are assembled together. FIG. 17 illustrates theassembled layers from FIG. 16. In FIG. 17, each layer can be readilyidentified as the corresponding layer of FIG. 16, assembled in the sameorder. During filling of the microfluidic channels in FIG. 17, thereagents are introduced at the top (1610), and continue to fill thechannels below. The liquid reagents are stopped at the stopper, which ispermeable to air and water vapor but impermeable to liquid water.Therefore, the air previously present in the channels is displaced bythe liquid and exits through the bottom of the device, which is exposedto air to allow evacuation of the gas. With this method, the overlay isfilled with reagents without presence of air. Two supporting layers(1640) allow compression of the other layers through the alignment rods.These layers allow passage of air in the bottom channels, and ofreagents in the top input, for example through openings.

FIG. 18 illustrates how the structure of FIG. 17 is filled withreagents. For example, a steel tube (1825) can be inserted in the inputchannel and connected to a pipe, such as a tygon line (1805), a lure-tubadapted (1810), and a syringe (1820) filled with barcodes and reagents(1815) such as PCR reagents for NGS library generation.

Subsequently, the overlay layers can be separated from the remaininglayers, as visible in FIG. 19, (1905, 1910). The liquids inside eachoverlay layer are held inside the wells by surface tension. Two overlaylayers (1905, 1910), each fabricated as described above, can be joinedtogether in order to obtain a unique two-dimensional mapping. Oneoverlay layer contains the x axis barcodes, and the other overlay layercontains the y axis barcodes. The two overlays, therefore, when alignedand assembled, form a unique combination of two barcodes (plus thereagents) in each well of the complete overlay, the complete overlaycomprising two overlay layers.

The completed overlay (1905 plus 1910) is attached to the tissue on atissue slide (1915), and then sealed on the top by a sealing slide(1920), which prevents any liquid leaking from one well to another. Theguide rods and frame (1925) can be joined with the other layers to forman overlay-tissue assembly (1930). Upon assembly, the xy barcodes andreagents inside each of the two overlay layers will diffuse and mix(1935), e.g. forming the complete respective library generation reagentmixture in each well.

FIG. 20 illustrates an exemplary device for analyzing tissues slideswith an attached overlay. For example, the device may have a lid (2005)that closes over the tissue slides, controls (2010), and housings for aplurality of tissue slides (2015). In some embodiments, the FFPE slidesto be analyzed are H&E stained, according to standard protocols. TheFFPE slides are placed in the device for imaging. The lid may containimaging devices, such as light sources and detectors. The bottom maycontain a temperature control device, such as a heater and cooler, forexample a Peltier heater and cooler.

The device of FIG. 20 is a thermal cycler and fluorescence imagerscanner that can detect and analyze each well in the overlay that isplaced over a tissue slide. Sequencing can be carried out before orafter scanning. The person of ordinary skill in the art will understandthat thermal cycling and imaging are carried out as a PCR method isapplied. For example, scanning can detect whether the PCR reactions havetaken place by receiving a signal from each well. In other embodiments,fluorescence scanning can take place after each thermal cycle. Slidesare thermally cycled to create an NGS library used in or for sequencing.

The NGS library can be collected by ‘holding’ the tissue slide with theoverlay and gently centrifugating it into a purpose-build collectionarea. Since the gene amplicons from each well in the overlay are markedwith a unique barcode, the amplicons from all wells can be collected ina single container. The information collected by measuring all thecellular material in the collected solution can be separated andattributed to each well, to form a two-dimensional map, due to thebarcode markers.

FIG. 21 illustrates an exemplary slide holder (2110) designed to providesupport to the slide (2125) and the overlay (2120) while they arecentrifuged. The slide holder keeps the overlay in place, and providesan area (2115) for the liquid to collect. FIG. 21 is a side viewcross-section of the slide holder (2110) that is spun in a centrifuge.The direction of the centrifugal force is shown by the arrow (2105). Thedirection of the gravitational force is shown by the arrow (2106). Whenthe slide (2125) is placed in the holder (2110), and spun in acentrifugem the liquid in the overlay (after removal or disruption ofthe cover on the overlay) will collect in the collection area (2115). Asthe centrifuge slows down gravity will dominate and the liquid willcollect in the area (2115). It can then be easily collected by a pipetteor similar device.

In other embodiments, centrifugation is not carried out, and othertechniques are used to collect the DNA. For example, after optionallypeeling off the overlay, chemical digestion or a combination ofdigestion and centrifugation could be used to collect all the DNA withthe attached barcodes. After collection of the DNA, a purification stepcan be carried out. The collected DNA can also be referred to as anamplicon, that is a piece of DNA or RNA that is the source or product ofthe amplification or replication event.

FIG. 22 illustrates an approach on how to prepare barcodes. A barcodeforward primer (2205) can be attached to a gene forward primer (2210) onone strand of the genomic DNA (2225), while a barcode reverse primer(2215) can be attached to a gene reverse primer (2220) on one strand ofthe genomic DNA (2225). The gene primers can combine into panels, whilethe barcode primers can be generic for any panel.

In the initial round of PCR, only gene primers (2210) (gene forwardprimer) and (2215) (gene reverse primer) are actively amplifying. Theseprimers have a region at the 3′ end designed to be complementary to aspecific target gene sequence (gene of interest), and a 5′ region thatis a ‘bridge’ sequence. In this embodiment, there are two differentbridge sequences for the gene primers: a first bridge sequence at a 5′region of the gene forward primer and a second bridge sequence at a 5′region of the gene reverse primer. In FIG. 22, the 3′ end of the geneprimers is shown parallel to the genomic DNA target (2225).

In the second round of PCR the products of the first round areamplified, so the reverse complements of the ‘bridge sequences’ of thegene primers are created. Therefore, after the second round of PCR, theDNA with the reverse complement of the ‘bridge’ sequences becomes atarget for the (2205) and (2220) barcode primers (via the ‘bridge’sequences at the 3′ ends of the barcode primers). In this embodiment,there are also two different bridge sequences for the barcode primers: afirst sequence at the 3′ end of the barcode forward primer identical tothe first bridge sequence of the gene forward primer and a second bridgesequence at the 3′ end of the barcode reverse primer identical to thesecond bridge sequence of the gene reverse primer. As a result, the geneprimers bind to and amplify the target gene sequence through the 3′ endthat is complementary to the target and the barcode primers amplify theamplification product of the gene primers (i.e. amplicon) through the 3′end bridge sequences of the barcode primers that are complementary tothe amplification product of the gene primers.

On their 5′ end, barcode primers (2205, 2220) encode the barcodesequence plus additional sequences 5′ of that. For instance, inreference to FIG. 14 and FIG. 22, it is possible to consider that theA,B,C . . . barcodes, that is (1405,1410,1425), are encoded in a seriesof (2205) barcode primers, and 1,2,3 . . . barcodes, that is(1415,1420,1430), are encoded in a series of (2220) barcode primers.

The term “complementary” as used herein refers to a property of singlestranded polynucleotides in which the sequence of the nucleotides on onestrand chemically matches the sequence on another strand to form adouble stranded polynucleotide. Chemically matching indicates that thenucleobases of the nucleotides in one strand can be non-covalentlyconnected via two or three hydrogen bonds with corresponding nucleobasesin another strand.

The person of ordinary skill in the art will understand that some of thesequences above are not described in detail (e.g. the ‘bridge’sequences). These sequences are required for and enable the sequencing.For instance, in some embodiments, the ‘bridge’ sequences correspond tosequencing primer sequences and the 5′ most sequence of (2205) and(2220) are sequences required to attach the library to the sequencingdevice. In principle, it is possible to use one long primer, instead oftwo shorter primers as described above. However, there are advantages tothe method described herein with two short primers. The longer primers(e.g. about 100 base pairs) that could be used as single primers tosubstitute the two short primers are very expensive to synthesize,several fold more expensive than two shorter primers. Additionally,primers that are very long (100 base pairs) tend not to work very wellin PCR. The approach described herein also separates the ‘barcodegeneration’ from ‘target amplification’. Thus, it makes it easier todesign a new library. It is also possible to use a ‘common’ pool of(2205,2220) primers with unique libraries of (2210,2215) primers, whichcan simplify the design. In other words, the embodiment of FIG. 22utilizes two short primers for each side, that is a total of four shortprimers. In other embodiments, a single long primer could be used foreach side, for a total of two long primers. However, the embodiment withshort primers has advantages as described above. The primers act asbarcodes that enable the two-dimensional positioning of the DNA strandto which the primers are attached to. Since each well of the overlay hasa unique combination of two barcodes, the DNA strands in each overlaywell can be uniquely identified and assigned to its barcoded spatialposition in the overlay.

In other embodiments, it is possible to use Uracil in place of Thymidinein the (2210, 2215) gene primers. Then, if there is a need to do furtheramplification of the library after it has been collected, it is possibleto treat the sample with UDG (uracil DNA glycosylase—which removesuracils, and renders that DNA unamplifiable). Subsequently, it ispossible to amplify with primers corresponding to the 5′ region (nobarcode) of the (2210, 2215) gene primers. This UDG treatment willensure that no ‘new’ barcode sequences are added to any amplificationproducts through addition by the (2210,2215) gene primers (which are noweffectively destroyed).

Sequencing can be performed on a commercial sequencer. Primer sequencesused during amplification can to be customized to the particularsequencer being used. Morphogenomic analysis can be carried out with thedevices described in the present disclosure. The placement of theoverlay wells over the tissue slides can be automatically determinedbased on the images of the slide before and after the overlay is placedon it. The genomic data for each well is known based on the barcodepairs from the sequencing. This information is ‘combined’ to enable, forexample, false coloring of the FFPE image based on genomic data.Morphometric parameters determined by software (or manually), can becorrelated with and/or visualized by, the software.

For example, integration of genomic and morphological information canreveal tumor heterogeneity. Cancer treatment decisions today arepredominantly based upon decades-old histological methods, which morerecently have been combined with simple phenotypic or molecular tests.To advance this paradigm, the present disclosure describes a digitalmolecular morphology platform that integrates histological images withnext-generation sequencing (NGS) or PCR data. This method allows thevisualization of DNA mutations and RNA expression levels across ahistological image, and the correlation of mutational or expressionstatus with cell morphological features. In this way, it is possible tomap tumor heterogeneity. The basic approach is to overlay amicrostructure on top of H&E-stained FFPE slides to create a largenumber of tissue-bottomed micro-wells, which can be as small as a singlecell or contain clusters of cells. Reagents are added to the micro-wellsand biochemical reactions are carried out and analyzed in place (PCR) oroffline (NGS). In the case of NGS, barcodes are used to reference theresulting sequence back to the tissue image. Software is then used toanalyze and visualize the resulting genetic information in the contextof the original histological image. In some embodiments, FETE cancerbiopsies using targeted cancer genes and gene panels can be used. Forexample, this approach can be used to measure and visualize KRAS G12Vmutational status in colorectal cancer tissues, using both PCR and NGSdetection modalities. With the method of the present disclosure, it ispossible to efficiently extract DNA from the tissue while maintaining aleak-proof physical integrity from one micro-well to another. Further,it is possible to PCR-amplify DNA in the micro-wells for directfluorescence detection, or to create libraries for sequencing. Detectionof KRAS G12V correlates well with malignant morphological features inthe H&E stained tissue, and appears to present a more sensitive way todetect mutations than PCR or NGS from bulk FFPE tissue alone. Themethods and devices of the present disclosure can be applied to avariety of research and clinical applications, ranging from assessmentof tumor heterogeneity and evolution, to prediction of patient outcome,to post-operative NGS-based characterization of surgical margins. Thisintegration of cellular and molecular data can more fully enableprecision medicine to guide the course of treatment and improveindividual patient outcomes.

In some embodiments, the microfluidics channel in a distributor chip,with the channel having n end wells, splits n−1 times. Each channel, itsinput port and its plurality of exit wells runs substantially parallelto either a previous or subsequent channel, depending on the position ofthe channel in the distributor chip.

The embodiments and examples set forth above are provided to give thoseof ordinary skill in the art a complete disclosure and description ofhow to make and use the embodiments of the devices, systems and methodsof the disclosure, and are not intended to limit the scope of what theinventors regard as their disclosure.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the arrangements, devices, compositions,systems and methods of the disclosure, and are not intended to limit thescope of what the inventors regard as their disclosure. All patents andpublications mentioned in the specification are indicative of the levelsof skill of those skilled in the art to which the disclosure pertains.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background, Summary, Detailed Description, andExamples is hereby incorporated herein by reference. All referencescited in this disclosure are incorporated by reference to the sameextent as if each reference had been incorporated by reference in itsentirety individually. However, if any inconsistency arises between acited reference and the present disclosure, the present disclosure takesprecedence.

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe disclosure claimed. Thus, it should be understood that although thedisclosure has been specifically disclosed by preferred embodiments,exemplary embodiments and optional features, modification and variationof the concepts herein disclosed can be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this disclosure as defined by the appended claims.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. As used in this specification and the appended claims,the singular forms “a,” “an,” and “the” include plural referents unlessthe content clearly dictates otherwise. The term “plurality” includestwo or more referents unless the content clearly dictates otherwise.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure pertains.

When a Markush group or other grouping is used herein, all individualmembers of the group and all combinations and possible subcombinationsof the group are intended to be individually included in the disclosure.Every combination of components or materials described or exemplifiedherein can be used to practice the disclosure, unless otherwise stated.One of ordinary skill in the art will appreciate that methods, deviceelements, and materials other than those specifically exemplified can beemployed in the practice of the disclosure without resort to undueexperimentation. All art-known functional equivalents, of any suchmethods, device elements, and materials are intended to be included inthis disclosure. Whenever a range is given in the specification, forexample, a temperature range, a frequency range, a time range, or acomposition range, all intermediate ranges and all subranges, as wellas, all individual values included in the ranges given are intended tobe included in the disclosure. Any one or more individual members of arange or group disclosed herein can be excluded from a claim of thisdisclosure. The disclosure illustratively described herein suitably canbe practiced in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein.

A number of embodiments of the disclosure have been described. Thespecific embodiments provided herein are examples of useful embodimentsof the disclosure and it will be apparent to one skilled in the art thatthe disclosure can be carried out using a large number of variations ofthe devices, device components, methods steps set forth in the presentdescription.

As will be obvious to one of skill in the art, methods and devicesuseful for the present methods can include a large number of optionalcomposition and processing elements and steps.

In particular, it will be understood that various modifications may bemade without departing from the spirit and scope of the presentdisclosure. Accordingly, other embodiments are within the scope of thefollowing claims.

The references in the present application, shown in the reference listbelow, are incorporated herein by reference in their entirety.

REFERENCES

-   [1] Shibata D, Hawes D, Li Z H, Hernandez A M, Spruck C H, Nichols    P W. Specific genetic analysis of microscopic tissue after selective    ultraviolet radiation fractionation and the polymerase chain    reaction. Am J Pathol (1992) 141:539-43.-   [2] Unger, M. A.; Chou, H. P.; Thorsen, T.; Scherer, A.;    Quake, S. R. Monolithic Microfabricated Valves and Pumps by    Multilayer Soft Lithography. Science 288, 113-116 (2000).-   [3] Kartalov, E P.; Walker, C.; Taylor, C. R.; Scherer, A.;    Anderson, W. F. Microfluidic Vias Enable Nested Bioarrays and    Autoregulatory Devices in Newtonian Fluids. Proc. Natl. Acad. Sci.    USA 103:33, 12280-12284 (2006).-   [4] Taylor, Becker, K. F. ‘Liquid Morphology’: Immunochemical    Analysis of Proteins extracted from Formalin Fixed Paraffin Embedded    Tissues: combining Proteomics with immunohistochemistry. Applied    Immunojhistochem Mol Morph 19, 1-9, 2011.-   [5] Shan-Rong Shi, Cheng Liu, Brian M. Balgley, Cheng Lee, and    Clive R. Taylor, Protein Extraction from Formalin-fixed,    Paraffin-embedded Tissue Sections: Quality Evaluation by Mass    Spectrometry. Journal of Histochemistry & Cytochemistry, Volume    54(6): 739-743, 2006.

What is claimed is:
 1. A method comprising: a) providing a distributorchip comprising: a plurality of microfluidics channels, eachmicrofluidics channel having an input port on a first surface and aplurality of output ports on a second surface opposite the firstsurface, each microfluidics channel, its input port and its plurality ofoutput ports laying on a plane parallel to a subsequent or previousmicrofluidics channel, thereby obtaining parallel rows of the outputports on the second surface; b) providing a first overlay comprisingparallel rows of microfluidics wells having positions corresponding tothe parallel rows of the output ports; c) providing a receiver chipcomprising parallel rows of microfluidics conduits having positionscorresponding to the parallel rows of the microfluidics wells of thefirst overlay; d) providing a stopper layer permeable to air andimpermeable to water; e) providing a support layer comprising parallelrows of openings corresponding to the parallel rows of the microfluidicsconduits of the receiver chip; f) positioning the distributor chip onthe first overlay, the first overlay on the receiver chip, the receiverchip on the stopper layer, and the stopper layer on the support layer;and g) aligning the distributor chip, the first overlay, the receiverchip, the stopper layer and the support layer, by aligning the parallelrows of the output ports, the parallel rows of the microfluidics wells,the parallel rows of the microfluidics conduits, and the parallel rowsof openings, thereby creating an aligned assembly.
 2. The method ofclaim 1, further comprising: h) inserting a solution in each input portof each microfluidics channel of the plurality of microfluidics channelsby pressuring the solution until air exits from the parallel rows ofmicrofluidics conduits of the receiver chip, through the stopper layer,into the parallel rows of openings of the support layer.
 3. The methodof claim 1, wherein each microfluidics channel binarily splits, betweenits input port and its plurality of output ports, a number of timesequal to the number of its output ports minus one.
 4. The method ofclaim 1, wherein providing a distributor chip is by photolithography or3D printing, and the distributor chip is made of a photocurable printedresin or polydimethylsiloxane.
 5. The method of claim 1, wherein the rowoverlay is made of silicone, silicon, or glass.
 6. The method of claim1, wherein the row overlay is made of pressure sensitive silicone, andproviding the row overlay comprises attaching a pressure sensitiveadhesive layer on at least one surface of the row overlay, and attachingat least one plastic foil layer on the at least one pressure sensitiveadhesive layer, the at least one plastic foil layer configured to bepeeled off to expose the pressure sensitive adhesive layer.
 7. Themethod of claim 1, wherein the stopper layer is made ofpolydimethylsiloxane.
 8. The method of claim 2, wherein the distributorchip, the first overlay, the receiver chip, the stopper layer and thesupport layer comprise alignment openings, and wherein aligning thedistributor chip, the first overlay, the receiver chip, the stopperlayer and the support layer comprises inserting alignment rods throughthe alignment openings.
 9. The method of claim 8, wherein each solutioninserted in each input port comprises same reagents and a differentbarcode fir each input port, and further comprising: j) removing thefirst overlay from the aligned assembly, thereby obtaining parallel rowsof microfluidics wells, each row having the same reagents and a uniquebarcode in each row of the parallel rows of microfluidics wells of thefirst overlay; k) providing a second overlay according to steps a)-j),wherein the solution for the second overlay comprises the same reagentsbut different barcodes than the solution for the first overlay; and l)positioning the second overlay on the first overlay, thereby obtainingan assembled overlay, comprising the first overlay and the secondoverlay, the assembled overlay comprising a plurality of assembledmicrofluidics wells, each assembled microfluidics well having the samereagents and a unique combination of two barcodes.
 10. The method ofclaim 1, wherein the microfluidics wells have a lateral dimensioncapable of holding liquids within by surface tension.
 11. The method ofclaim 9, herein the reagents are for polymerase charm reaction.
 12. Themethod of claim 9, further comprising: attaching the assembled overlayon a tissue slide; and thermally cycling the tissue slide.
 13. Themethod of claim 12, further comprising imaging the tissue slide.
 14. Themethod of claim 13, wherein the imaging is after each thermal cycle. 15.The method of claim 12, further comprising removing the solution fromthe assembled overlay.
 16. The method of claim 15, wherein the removingis by centrifugating.
 17. The method of claim 16, further comprisingcarrying out morphogenomic analysis for presence of gene mutations bysequencing of gene amplicons including sequences of the uniquecombinations of two barcodes.
 18. The method of claim 12, wherein thetissue slide is a formalin-fixed, paraffin-embedded tissue slide stainedwith hematoxylin and eosin.
 19. The method of claim 18, wherein theassembled overlay comprises at least two thousands wells, each wellhaving lateral dimensions between 5×5 μm and 1000×1000 μm.
 20. Themethod of claim 17, wherein carrying out morphogenomic analysiscomprises determining a gene frequency or a mutation frequency indifferent regions of the tissue slide corresponding to differentassembled microfluidics wells.
 21. The method of claim 16, furthercomprising creating a two-dimensional map of genetic information of thetissue slide by assigning the genetic information to each uniquecombination of two barcodes.